SV40 large T antigen (T Ag) is capable of transforming a variety of normal cells into cancer cells. Cellular transformation is dependent on T Ag's ability to interact with key cellular regulatory proteins including pRb and p53. This laboratory has recently cloned p185, a novel T Ag associated protein. Furthermore, we have identified a small region of T Ag that is required for binding to p185. Mutation of this region of T Ag eliminates p185 binding but does not affect T Ag's ability to bind and inactivate pRb and p53. T Ag mutants that fail to bind to p185 are unable to transform primary mouse embryo fibroblasts suggesting that T Ag binding to p185 is required for transformation. p185 has significant homology to the cullin family of E3 ubiquitin ligases and to Apc10/Doc1, a component of the anaphase-promoting complex (APC). Cullins and the APC promote the degradation of specific protein substrates by serving as a scaffold for protein substrates and E2 ubiquitin conjugating enzymes. Preliminary Studies supports the hypothesis that p185 is a novel E3 ubiquitin ligase. In addition to cloning p185, we have cloned two p185-associated proteins including p281, highly related to p185, and an F-box containing protein, Fbw6/Fbx29. These results suggest that T Ag binds specifically to a novel cullin with potential to act as an E3 ubiquitin ligase and that this interaction is critical for cellular transformation. This proposal seeks to determine how p185 contributes to T Ag-mediated transformation. In addition, this proposal seeks to identify additional binding proteins of p185, to characterize the p185 ubiquitinating activity, and to determine if p185 serves as a tumor suppressor in a knockout mouse model.